Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

Timothy J. Ragan, Duncan B. Ross, Malik M. Keshwani, Thomas K Harris

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D2ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D2ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.

Original languageEnglish
Pages (from-to)271-279
Number of pages9
JournalProtein Expression and Purification
Volume57
Issue number2
DOIs
StatePublished - Feb 1 2008

Fingerprint

Ribosomal Protein S6 Kinases
70-kDa Ribosomal Protein S6 Kinases
Phosphorylation
Purification
Phosphotransferases
Catalytic Domain
Peptides
Size exclusion chromatography
Protein-Serine-Threonine Kinases
Chemical shift
Gel Chromatography
Plasticity
Polyacrylamide Gel Electrophoresis
Proteins
Homeostasis
Chemical activation
Nuclear magnetic resonance
Amino Acids

Keywords

  • Autoinhibition
  • Custom gene synthesis
  • Disordered proteins
  • Intrinsic disorder
  • Minimal media
  • PDK1
  • Phosphoinositide-dependent protein kinase-1
  • Proteolysis inhibition

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{4078c0c0bb934aa0bb296f6425efba43,
title = "Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)",
abstract = "S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D2ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D2ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.",
keywords = "Autoinhibition, Custom gene synthesis, Disordered proteins, Intrinsic disorder, Minimal media, PDK1, Phosphoinositide-dependent protein kinase-1, Proteolysis inhibition",
author = "Ragan, {Timothy J.} and Ross, {Duncan B.} and Keshwani, {Malik M.} and Harris, {Thomas K}",
year = "2008",
month = "2",
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doi = "10.1016/j.pep.2007.09.014",
language = "English",
volume = "57",
pages = "271--279",
journal = "Protein Expression and Purification",
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TY - JOUR

T1 - Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

AU - Ragan, Timothy J.

AU - Ross, Duncan B.

AU - Keshwani, Malik M.

AU - Harris, Thomas K

PY - 2008/2/1

Y1 - 2008/2/1

N2 - S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D2ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D2ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.

AB - S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D2ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D2ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.

KW - Autoinhibition

KW - Custom gene synthesis

KW - Disordered proteins

KW - Intrinsic disorder

KW - Minimal media

KW - PDK1

KW - Phosphoinositide-dependent protein kinase-1

KW - Proteolysis inhibition

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U2 - 10.1016/j.pep.2007.09.014

DO - 10.1016/j.pep.2007.09.014

M3 - Article

C2 - 17980619

AN - SCOPUS:37349113787

VL - 57

SP - 271

EP - 279

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -